Although glutamate is thought to be the neurotransmitter at the invertebrate neuromuscular junction, acetylcholine is accepted as the primary neurotransmitter of the vertebrate motoneurons. N-acetylaspartylglutamate, a dipeptide localized in putative glutamatergic neurons in brain, is also found in high concentrations (> mM) in mammalian motoneurons and the ventral roots of spinal cord. N-acetylaspartylglutamate, which is released from neurons by depolarization in a Ca(2+)-dependent fashion, is implicated in glutamatergic transmission in two ways: it is a partial agonist at NMDA receptors, and it is cleaved to yield extracellular glutamate and N-acetylasparate by the specific peptidase N-acetylated alpha-linked acidic dipeptidase. Given the localization of N-acetylaspartylglutamate in motor neuronal perikarya and axons, we wondered whether N-acetylaspartylglutamate or glutamate cleaved from N-acetylaspartylglutamate by N-acetylated alpha-linked acidic dipeptidase may also play a role in neuromuscular transmission. Here we describe the immunocytochemical detection at the rat neuromuscular junction of N-acetylaspartylglutamate in terminals of motoneurons, of N-acetylated alpha-linked acidic dipeptidase in perisynaptic Schwann cells, and of the NMDAR-1 glutamate receptor subunit on postsynaptic muscle membranes. These results point to a potential role for N-acetylaspartylglutamate at the rat neuromuscular junction. Further, this is the first demonstration of a glutamate receptor protein at vertebrate neuromuscular synapses. Together with other recent findings, our results suggest that glutamate-like molecules are involved in neuromuscular transmission not only in invertebrates but also in veretebrates where they may modulate signaling by acetylcholine.

Photoactivated adenylate cyclase alpha (PACalpha) is a light-activated adenylate cyclase that was originally cloned from the eye spot of the protozoan Euglena gracilis. PACalpha has been shown to rapidly increase intracellular cyclic adenosine monophosphate (cAMP) in vivo in Xenopus oocytes and HEK293 cells, increase the spike width in Aplysia sensory neurons, and modify behavior in Drosophila. Using the GAL4 UAS system, we heterologously expressed PACalpha in motorneurons and quantified the effects of its activation at the neuromuscular junction of the Drosophila third instar wandering larva, a well-characterized model synapse. By recording from body-wall muscle 6, we show that the presynaptic activation of PACalpha with blue light significantly increased miniature excitatory junction potential (mEJP) frequency in the presence of calcium with a delay of about 1 minute. Similar effects have been observed in previous studies that utilized adenylate cyclase agonists (Forskolin) or membrane-permeable cAMP analogs [dibutyryl cAMP and 4-chlorophenylthio-(CPT)-cAMP] to increase presynaptic cAMP concentrations. PACalpha's efficacy in combination with its specificity make it an invaluable tool for the rapid regulation of cAMP in vivo and for investigating the mechanisms by which cAMP can modulate synaptic transmission and neuronal plasticity in Drosophila.

Evoked synaptic activity within the CNS and at the neuromuscular junction in most in vivo preparations studied occurs not with single isolated stimuli, but with trains, or bursts, of stimuli. Although for ease in studying the mechanisms of vesicular synaptic transmission one often uses single discrete stimuli, the true mechanisms in the animal may be far more complex. When repetitive stimuli are present at a nerve terminal, often a heightened (i.e., facilitated) postsynaptic potential can be as a result. Facilitation is commonly used as an index of synaptic function and plasticity induced by chronic stimulation or by neuromodulation. The mechanisms that give rise to facilitation are thought to be the same that may underlie short-term learning and memory [C.H. Bailey, E.R. Kandel, Structural changes accompanying memory storage. Annu. Rev. Physiol. 55 (1993) 397-426.]. Differences in short term facilitation (STF) are seen depending on the conventional stimulation paradigm (twin pulse, train, or continuous) used to induce facilitation. Thus, a battery of paradigms should be used to characterize synaptic function to obtain a closer understanding of the possible in vivo conditions.

The regulation of synaptic efficacy is essential for the proper functioning of neural circuits. If synaptic gain is set too high or too low, cells are either activated inappropriately or remain silent. There is extra complexity because synapses are not static, but form, retract, expand, strengthen, and weaken throughout life. Homeostatic regulatory mechanisms that control synaptic efficacy presumably exist to ensure that neurons remain functional within a meaningful physiological range. One of the best defined systems for analysis of the mechanisms that regulate synaptic efficacy is the neuromuscular junction. It has been shown, in organisms ranging from insects to humans, that changes in synaptic efficacy are tightly coupled to changes in muscle size during development. It has been proposed that a signal from muscle to motor neuron maintains this coupling. Here we show, by genetically manipulating muscle innervation, that there are two independent mechanisms by which muscle regulates synaptic efficacy at the terminals of single motor neurons. Increased muscle innervation results in a compensatory, target-specific decrease in presynaptic transmitter release, implying a retrograde regulation of presynaptic release. Decreased muscle innervation results in a compensatory increase in quantal size.